There is an increasing recognition that the medical significance of fungal infections has dramatically increased. There is a rising incidence of these infections as well as recognition that fungal species heretofore thought to be non-pathogenic are indeed a cause of disease. Presently, there are limited therapeutic options for the treatment of these infections and there is an urgent need for the development of additional effective and safe antifungal agents.
Human infections caused by fungi can be divided into four main groups depending on the location of the infection. These include 1) superficial infections of the outer layers of the skin, nails, and hair follicles and are usually caused by Dermatophytes and Candida species, 2) subcutaneous infections of deeper layers of the skin and subcutaneous tissues and are caused most commonly by Sporothrix schenckii and Pseudallescheria boydii, 3) mucosal infections of the gastrointestinal tract and the genitourinary tract and are usually caused by Candia species, and 4) systemic infections of the bloodstream and deep organs of the body which are caused by by an increasing number of fungal pathogens.
Systemic fungal infections, in particular, have become a significant medical problem. According to statistics from the Centers for Disease Control and Prevention, the incidence of systemic fungal infections, particularly those caused by Candida species have climbed dramatically over the last 20 years. Candida species are the fourth most common microorganisms isolated from the bloodstream of hospitalized patients in the United States. These infections are serious: despite antifungal therapy, the attributable mortality of Candida species bloodstream infections is 30–40%.
The majority of systemic fungal infections are caused by Candida albicans. Infections by non-albicans species such as Candida glabrata, Candida tropicalis, and Candida krusei have been increasing. Though rare, infections by Aspergillus species, Mucorales, and Fusarium have also been increasing. Predisposing factors that lead to these infections includes treatment with broad-spectrum antibacterial antibiotics, a compromised immune system caused by cancer chemotherapy, treatment for transplant rejection or treatment of autoimmune illnesses with corticosteroids, and underlying illnesses such as HIV infection. Additionally elderly and debilitated persons are at risk for these infections.
The diagnosis of fungal infections is often difficult. Many opportunistic fungal infections cannot be diagnosed by routine blood or tissue culture and must be treated empirically in severely immune-compromised patients (Walsh et al. (199 1) Rev. Infect. Dis. 13:496).
At present, there is a limited number of therapeutics available to treat these infections. Therapy is further complicated by the fact that the available therapeutic agents are associated with severe toxicity and the need for intravenous access. The polyene antifungal agent, amphotericin B, has served as the cornerstone of therapy for the several decades. It is administered intravenously and is associated with fever and chills during its administration, renal insufficiency and anemia. Recently, newer lipid formulations of amphotericin B have become available and are associated with a lesser incidence of adverse effects. Azole antifungal agents are the othermajor class of antifungal therapeutics that are available for the treatment of fungal infections. Unlike amphotericin B, these agents are administered both intravenously and orally. In general, these agents are not considered as potent as amphotericin B and are usually used in less severe infections. They have also been associated with hepatic dysfunction. Another recent problem that has been noted is the increasing incidence of resistance to the azole antifungal agents.
The development of effective and safe treatment of fungal infections has lagged behind the therapy for bacterial infections. There are numerous commentators who have speculated on this apparent neglect. See, for example, Georgopapadakou et al. (1994) Science 264:371. First, it is difficult. Like mammalian cells, fungi are eukaryotes and thus agents that inhibit fungal protein, RNA, or DNA biosynthesis may do the same in the patient's own cells, producing toxic side effects and the attendant difficulty administering this agent to a patient. Second, life-threatening fungal infections were thought, until recently, to be too infrequent to warrant aggressive research by the pharmaceutical industry. Finally, because pathogenic fungi are difficult to culture, and because many of them do not reproduce sexually, microbiological and genetic research into the disease-causing organisms has lagged far behind research into other organisms.
The progression of a proliferating eukaryotic cell through the cell-cycle checkpoints is controlled by an array of regulatory proteins that guarantee that mitosis occurs at the appropriate time. Protein phosphorylation is the most common post-translational modification that regulates processes inside the cells, and a large number of cell cycle transitions are regulated by, in addition to protein-protein interactions, the phosphorylation states of various proteins. In particular, the execution of various stages of the cell-cycle is generally believed to be under the control of a large number of mutually antagonistic kinases and phosphatases.
A paradigm for these controls is the CDC2 protein kinase, a cyclin-dependent kinase (CDK) whose activity is required for the triggering of mitosis in eukaryotic cells (for reviews, see Hunt (1989) Curr. Opin. Cell Biol. 1:268–274; Lewin (1990) Cell 61:743–752; and Nurse (1990) Nature 344:503–508). During mitosis, the CDC2 kinase appears to trigger a cascade of downstream mitotic phenomena such as metaphase alignment of chromosomes. segregation of sister chromatids in anaphase, and cleavage furrow formation. Many target proteins involved in mitotic entry of the proliferating cell are directly phosphorylated by the CDC2 kinase. For instance, the CDC2 protein kinase acts by phosphorylating a wide variety of mitotic substrates involved in regulating the cytoskeleton of cells, such that entry into mitosis is coordinated with dramatic rearrangement of cytoskeletal elements.
The CDC2 kinase is subject to multiple levels of control. One well-characterized mechanism regulating the activity of CDC2 involves the phosphorylation of tyrosine, threonine and serine residues, the phosphorylation level of which varies during the cell-cycle (Krekk et al. (1991) EMBO J. 10:305–316; Draetta et al. (1988) Nature 336:738–744; Dunphy et al. (1989) Cell 58:181–191; Morla et al. (1989) Cell 58:193–20–3); Gould et al. (1989) Nature 342:39–45; and Solomon et al. (1990) Cell 63:1013–1024).
The phosphorylation of CDC2 on Tyr-15 and Thr-14, two residues located in the putative ATP binding site of the kinase, negatively regulates kinase activity. This inhibitory phosphorylation of CDC2 is mediated at least in part by the weel and mikI tyrosine kinases (Russel et al. (1987) Cell 49:559–567; Lundgren et al. (1991) Cell 64:1111–1122; Featherstone et al. (1991) Nature 349:808–811; and Parker et al. (1992) PNAS 89:2917–2921). These kinases act as mitotic inhibitors, over-expression of which causes cells to arrest in the G2 phase of the cell-cycle. By contrast, loss of function of weel causes a modest advancement of mitosis, whereas loss of both weel and mikI function causes grossly premature mitosis,. uncoupled from all checkpoints that normally restrain cell division (Lundgren et al. (199 1) Cell 64:1111–1122).
As the cell is about to reach the end of G2, dephosphorylation of the CDC2- inactivating Thr-14 and Tyr-15 residues occurs leading to activation of the CDC2 complex as a kinase. A stimulatory phosphatase, known as CDC25, is responsible for Tyr-15 and Thr-14 dephosphorylation and serves as a rate-limiting mitotic activator. (Dunphy et al. (1991) Cell 67:189–196, Lee et al. (1992) Mol. Biol. Cell. 3:73–84; Millar et al. (1991) EMBO J :4301–4309; and Russell et al. (1986) Cell 45:145–153).
Recent evidence indicates that both the CDC25 phosphatase and the CDC2-specific tyrosine kinases are detectably active during interphase, suggesting that there is an ongoing competition between these two activities prior to mitosis (Kumagai et al. (1992) Cell 70:139–151; Smythe et al. (1992) Cell 68:787–797; and Solomon et al. (1990) Cell 63:1013–1024). This situation implies that the initial decision to enter mitosis involves a modulation of the equilibrium of the phosphorylation state of CDC2 at these residues, which is likely controlled by variation of the rate of tyrosine dephosphorylation of CDC2 and/or a decrease in the rate of its tyrosine phosphorylation.
In addition to the inhibitory phosphorylation of Cdks, most Cdks also require binding of a cyclin and phosphorylation of a threonine (residue 169 in S cerevisiae p34cdc2). This phosphorylation is carried out by CAK, the“Cdk-activating kinase”. The cerevisiae CAKI binds tightly to and phosphorylates Cdc28, thereby allowing its subsequent activation by the binding of a cyclin. The CAKI gene is essential for yeast cell viability, and Cdc28 phosphorylation and activity are conditionally inhibited in a CAKI temperature-sensitive mutant. For instance, CAKI is required for vegetative growth and spore wall morphogenesis (see Wagner et al. (1997) EMBO J. 16:1305; and Kaldis (1999) Cell Mol Life Sci 55:284).
In vertebrates, CAK is a trimeric enzyme containing CDK7, cyclin H, and MATI. CAK from the budding yeast was identified as an unusual 44-kilodalton protein kinase, CAKI, that is only distantly related to CDKs. CAKI accounted for most CAK activity in yeast cell lysates, and its activity was constant throughout the cell cycle. The CAK I gene was essential for cell viability. Thus, the major CAK in S. cerevisiae is distinct from the vertebrate enzyme, suggesting that budding yeast and vertebrates may have evolved different mechanisms of CDK activation.
Apart from phosphorylation, the regulation of the Cdc2-cyclin B complex involves a small ancillary subunit called p9sucl, a member of the Sucl/Cks family of proteins. Although p9 is not required for the catalytic activity of Cdc2, it appears to be responsible for the interaction of MPF with some of its critical regulators. For example, Xenopus egg extracts from which p9 has been completely removed by immunodepletion cannot undergo cell-cycle transitions normally. Specifically, mitotic egg extracts lacking p9 cannot exit mitosis properly due to a defect in the ubiquitin-mediated proteolysis of cyclin B. A molecular description of how p9 helps to mediate the exit from mitosis should provide valuable insights into Cdk regulation.
In the past decade, however, more antifungal drugs have become available. Nevertheless there are still major weaknesses in their spectra, potency, safety, and pharmacokinetic properties, and accordingly it is desirable to improve the panel of anti-fungal agents available to the practitioner.